The gamma-carboxyglutamic acid domain of human factor VIIa is essential for its interaction with cell surface tissue factor.
Previous studies indicated that both plasma-derived and recombinant human factor VIIa specifically interacted with tissue factor on the surface of a human bladder carcinoma cell line (J82). In the presence of calcium ions, factor VIIa interacted with approximately 300,000 binding sites/cell with a dissociation constant (Kd) of 3.25 nM (Sakai, T., Lund-Hansen, T., Paborsky, L., Pedersen, A. H., and Kisiel, W. (1989) J. Biol. Chem. 264, 9980-9988). In this study, we compare recombinant human factor VIIa and a preparation of recombinant human factor VIIa lacking the gamma-carboxyglutamic acid domain (GD-rVIIa) with respect to their interaction with J82 cell surface tissue factor. Interaction of GD-rVIIa with J82 monolayers at 37 degrees C was specific, saturable, and exhibited a hyperbolic profile. Scatchard plots of the binding data obtained at 37 degrees C indicated a single class of binding sites for GD-rVIIa with a Kd value of 2.5 nM. GD-rVIIa interacted with about 10,000 binding sites/cell. In contrast to the tissue factor-specific binding observed for intact factor VIIa, specific binding of GD-rVIIa to the J82 cell surface was neither influenced by calcium nor blocked by prior incubation of the cells with polyclonal anti-tissue factor apoprotein IgG. In addition, cell-bound GD-rVIIa failed to activate human factor X. These results indicate that the gamma-carboxyglutamic acid domain of factor VIIa is essential for its interaction with cell surface tissue factor.
Sakai
T
T
Blood Systems Research Foundation Laboratory, Department of Pathology, University of New Mexico School of Medicine, Albuquerque 87131.
Lund-Hansen
T
T
Thim
L
L
Kisiel
W
W
D015055:1-Carboxyglutamic Acid; D000595:Amino Acid Sequence; D001059:Apoproteins / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D015942:Factor VIIa / Q000378:metabolism*; D005170:Factor X / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D001749:Urinary Bladder Neoplasms
D001059:Apoproteins; D011994:Recombinant Proteins; D015055:1-Carboxyglutamic Acid; D005170:Factor X; D015942:Factor VIIa; D002118:Calcium